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Molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal
haemolytic anaemia, concomitant infection
Sándor Hornok, Marina L. Meli, András Erdös, István Hajtós, Hans Lutz, Regina Hofmann-Lehmann
To cite this version:
Sándor Hornok, Marina L. Meli, András Erdös, István Hajtós, Hans Lutz, et al.. Molecular char- acterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia, concomitant infection. Veterinary Microbiology, Elsevier, 2009, 136 (3-4), pp.372.
�10.1016/j.vetmic.2008.10.031�. �hal-00532529�
Accepted Manuscript
Title: Molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia, concomitantAnaplasma ovisinfection Authors: S´andor Hornok, Marina L. Meli, Andr´as Erd˝os, Istv´an Hajt´os, Hans Lutz, Regina Hofmann-Lehmann
PII: S0378-1135(08)00506-3
DOI: doi:10.1016/j.vetmic.2008.10.031
Reference: VETMIC 4259
To appear in: VETMIC Received date: 3-8-2008 Revised date: 27-10-2008 Accepted date: 29-10-2008
Please cite this article as: Hornok, S., Meli, M.L., Erd˝os, A., Hajt´os, I., Lutz, H., Hofmann-Lehmann, R., Molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia, concomitant Anaplasma ovis infection, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2008.10.031
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Accepted Manuscript
Molecular characterization of two different strains of haemotropic
1
mycoplasmas from a sheep flock with fatal haemolytic anaemia and
2
concomitant Anaplasma ovis infection
3 4
Sándor Hornok,
a* Marina L. Meli,
bAndrás Erdős,
cIstván Hajtós,
c5
Hans Lutz,
band Regina Hofmann-Lehmann
b6 7
a
Department of Parasitology and Zoology, 8
Faculty of Veterinary Science, Szent István University, Budapest, Hungary 9
10
b
Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland 11
12
c
County Veterinary Station, Borsod-Abaúj-Zemplén, Miskolc, Hungary 13
14 15
* Corresponding author. Mailing address: Department of Parasitology and Zoology, Faculty 16
of Veterinary Science, Szent István University, István u. 2., 1078 Budapest, Hungary. Phone:
17
(36 1) 478 4187, Fax: (36 1) 478 4193. E-mail: Hornok.Sandor@aotk.szie.hu 18
19
Abstract
20
After the first outbreak of fatal Mycoplasma ovis infection (eperythrozoonosis) in a sheep 21
flock in Hungary (1997), a second wave of the disease was noted in 2006, with different 22
seasonal pattern and affected age group, as well as increased mortality (5.5%). The aim of the 23
present study was to molecularly characterize the causative agent and to reveal underlying 24
Manuscript
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factors of the second wave of the disease. Remarkably, among the 33 sheep examined, 17 25
were infected with two strains of haemotropic mycoplasmas. Cloning and sequencing isolates 26
of the latter showed that one of the strains was 99.4-99.8% identical to M. ovis (AF338268), 27
while the second was only 96.8-97.9% identical and contained a 17-bp deletion. Different 28
isolates of both strains were demonstrated in the same animal. When analyzing possible risk 29
factors for fatal disease outcome, we found that among sheep born prior to the 1997 outbreak 30
significantly more animals survived the second outbreak than succumbed to disease. In 31
addition, locally born sheep were less frequently diseased than sheep introduced into the flock 32
from other places. This suggests an immunoprotective effect in some animals. Concurrent 33
infection with Anaplasma ovis was detected in 24 of the 33 evaluated sheep. In conclusion, 34
this is the first study to demonstrate the existence of and characterize two genetically distinct 35
ovine haemotropic mycoplasma strains in a sheep flock with fatal haemolytic anaemia.
36 37
Keywords: sheep; anaemia; haemotropic mycoplasmas; Mycoplasma ovis; Anaplasma ovis 38
39
1. Introduction
40 41
Haemotropic mycoplasmas are unculturable bacteria that may cause haematological 42
disorders in various hosts (Ristic and Kreier, 1984). They had been classified as members of 43
the Anaplasmataceae family (order Rickettsiales) and were further divided into two genera:
44
Haemobartonella, characterized by mostly epierythrocytic, coccoid forms, and 45
Eperythrozoon, existing mostly in ring forms on red blood cells and free in the plasma.
46
However, analysis of 16S rRNA gene sequences revealed the close relationship of these 47
bacteria with the genus Mycoplasma (Neimark et al., 2001). Until now, the only species 48
recognized in sheep was Mycoplasma ovis (formerly Eperythrozoon ovis), which may induce
49
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poor weight gain, severe anaemia, and even mortality in lambs and, occasionally, young adult 50
sheep (Campbell et al., 1971; Daddow, 1979). Mild clinical signs and persistent bacteraemia 51
associated with chronic infection have also been described in older animals (Gulland et al., 52
1987; Mason and Statham, 1991).
53
In Hungary, the first and thus far only reported case of ovine eperythrozoonosis 54
resulted in 8.5% (35 out of 412) morbidity and 2.2% (9 out of 412) mortality (Hajtós et al., 55
1998). The disease manifested between August and November of 1997, and mainly affected 56
yearlings. Mycoplasma ovis infection apparently persisted in the same flock of sheep, with 57
only sporadic and mild pathogenic effects for several years. However, in July and August of 58
2006, a new disease outbreak occurred. The aim of the present study was to characterize the 59
causative agent of the latter using molecular methods and to reveal underlying factors that 60
may have contributed to the more severe outcomes, as well as to the altered seasonal pattern 61
and affected age group of the disease.
62 63
2. Materials and methods
64 65
2.1. Case history and sample collection 66
67
Clinical signs of fatal M. ovis infection (anaemia, submandibular oedema, weight loss) were 68
noted between July and August of 2006 in 39 animals from a flock of 546 sheep (designated 69
flock A) in Northeast Hungary. Data obtained for the animals, including the 30 sheep that 70
died during the outbreak (group A1), were recorded. Ten months after the first clinical 71
manifestation, EDTA anticoagulated blood samples were collected by jugular venipuncture 72
from a total of 33 sheep: 9 sheep with typical clinical signs that survived the outbreak (group 73
A2); 16 animals with no characteristic clinical signs (group A3); and 8 animals from a
74
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neighboring flock (designated flock B) in which clinical signs attributable to M. ovis had 75
never been observed. Blood smears were prepared from fresh EDTA anticoagulated blood and 76
stained with Giemsa. Haematological values were determined using an Abacus haematology 77
analyzer (Diatron GmbH, Vienna, Austria). Sheep in both flocks (A and B) were supervised 78
by the same veterinarian and received the same nutrition and prophylactic medication, but 79
were housed in separate buildings on the same farm and grazed in different pastures.
80 81
2.2. Nucleic acid extraction 82
83
DNA was purified from 33 blood samples using the MagNA Pure LC total nucleic acid 84
isolation kit (Roche Diagnostics, Rotkreuz, Switzerland). First, 100
l of EDTA85
anticoagulated blood was diluted in the same volume of PBS without MgCl
2and CaCl
286
(Invitrogen, Basel, Switzerland). Then, 300
l of lysis buffer containing guanidinium87
thiocyanate and Triton X-100 (Roche Diagnostics) was added. The lysed samples were loaded 88
into wells on the automated MagNA Pure LC Instrument. Two PBS controls were included in 89
each run to monitor potential cross-contamination. The elution volume was 100
l. Samples90
were stored at -20C until further analysis. The presence of amplifiable DNA was confirmed 91
for each sample using an 18S rRNA gene real-time TaqMan PCR assay (Applied Biosystems, 92
Rotkreuz, Switzerland) (data not shown).
93 94
2.3. Real-time TaqMan PCR to detect M. ovis infection 95
96
A quantitative real-time TaqMan PCR assay developed for M. wenyonii (AF016546) was 97
used to screen the DNA samples for M. ovis (Meli et al., submitted for publication). The PCR 98
amplified a 119-bp long fragment of the 16S rRNA gene of M. wenyonii. Primer and probe
99
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sequences (forward primer, 5’-CCA CGT GAA CGA TGA AGG TCT T-3’; reverse primer, 100
5’-GGC ACA TAG TTA GCT GTC ACT TAT TCA A-3’; and probe, 5’-6FAM-AGT ACC 101
ATC AAC GCG CGC TGA TTT CCT AG-MGB-3’) demonstrated 100% identity with the 102
sequence of the 16s rRNA gene of M. ovis (AF338268). The 25
l PCR reaction mixture103
contained 900 nM of each primer, 0.25
M probe, 12.5 l of 2qPCR MasterMix 104
(Eurogentec, Seraing, Belgium), and of 5
l template DNA. The amplification mixture105
contained dUTP for use with uracil-N-glycosylase to prevent carryover of the PCR 106
amplicons. The cycling conditions were: incubation for 2 min at 50C and then a 10 min 107
initial denaturation at 95C, followed by 45 cycles of 15 s at 95C and 1 min at 60C using 108
the ABI Prism 7700 sequence detection system (Applied Biosystems).
109 110
2.4. Amplification and sequencing of the 16S rRNA gene of ovine haemotropic 111
mycoplasmas 112
113
Initially, a conventional PCR was used to amplify an approximately 200-bp long region of the 114
16S rRNA gene as described elsewhere (Jensen et al., 2001). PCR products were visualized 115
with ethidium bromide in a 2.5% agarose gel.
116
In a second step, two samples from each group (A2, A3, and B) were chosen for 117
further analysis by cloning and sequencing. Samples were selected according to Ct values 118
(low Ct value = high load), and assessed by conventional PCR using species-specific primers 119
for M. ovis (forward: 5’-AGAGTT TGA TC(A/C) TGG CTC AG-3’, reverse: 5’-CGG TTA 120
CCT TGT TAC GAC TT-3’), which amplify a 1,485-bp region of the 16S rRNA gene, as 121
described elsewhere (20). The 25 l reaction mixture contained 5 l of 5 High Fidelity PCR 122
buffer (Finnzymes: BioConcept, Allschwil, Switzerland), 0.5
M of each primer, 0.2 mM123
dNTP mixture (Sigma-Aldrich, Buchs, Switzerland), 1 U of Phusion High Fidelity DNA
124
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polymerase (Finnzymes), 15.25
l of nuclease-free water, and 2.5 l of template DNA.125
Amplification was performed using a T-personal 48 Thermocycler (Biometra GmbH, 126
Goettingen, Germany). The thermal profile consisted of an initial denaturation step at 98C 127
for 3 min, followed by 35 cycles of 10 s at 98C, 30 s at 60C, and 1 min at 72C, with a final 128
elongation at 72C for 10 min. A portion of the PCR product was visualized in a 1% agarose 129
gel stained with ethidium bromide after electrophoresis. The remaining portion was purified 130
with the GenElute PCR Clean-Up Kit (Sigma-Aldrich) and cloned into the pCR®4-TOPO®
131
(Invitrogen) vector using the TOPO TA Cloning® Kit for Sequencing (Invitrogen). Clones 132
were screened by colony PCR, i.e. the M. wenyonii TaqMan assay as described above. The 133
fifteen positive clones that produced the lowest Ct values were selected, and plasmid DNA 134
was purified using the QIAprep Spin Miniprep kit (Qiagen, Hombrechtikon, Switzerland).
135
Cycle sequencing was performed with 4 l of DNA, 3.3 pmol of plasmid-specific primers, an 136
additional internal primer (5’-GGG AGG CTG ATC CAT TGT TA-3’), and the BigDye 137
Terminator Cycle Sequencing Ready Reaction Kit v1.1 (Applied Biosystems). The cycling 138
conditions were as follows: 1 min at 96°C, followed by 25 cycles of 96°C for 10 s and 50°C 139
for 5 sec, and a final incubation at 60°C for 4 min. Products were purified using SigmaSpin
140
Post-Reaction Purification Columns (Sigma-Aldrich) and analyzed on the ABI Prism 310 141
Genetic Analyzer (Applied Biosystems).
142
Obtained sequences were edited and aligned with a consensus sequence using 143
SeqScape (Version 1.1, Applied Biosystems), and then compared to the 16S rRNA gene of M.
144
ovis (AF338268). For phylogenetic analysis, the sequences were aligned with known 145
mycoplasma sequences from GenBank using ClustalW (Thompson et al., 1994) and, if 146
necessary, manually adjusted. Only the nucleotides available for all included sequences were 147
used in the phylogenetic analysis. A bootstrap phylogenetic tree demonstrating the 148
relationship of sheep isolates to other haemoplasma species was created by the Neighbor-
149
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Joining method (Saitou and Nei, 1987) using a distance matrix corrected for nucleotide 150
substitutions based on the Kimura 2-parameter model. The dataset was resampled 1,000 times 151
to generate bootstrap values. Phylogenetic and molecular evolutionary analyses were 152
conducted using MEGA version 4 (Kumar et al., 2004).
153
New sequences were submitted to GenBank (accession numbers: EU165509-165513, 154
EU828579-EU828582).
155 156
2.5. Evaluation of Anaplasma ovis infection 157
158
The blood samples were screened for the presence of members of the Anaplasmataceae family 159
by conventional PCR with the following primers: Ehr1 (5’- TTT ATC GCT ATT AGA TGA 160
GCC TAT G -3’) and Ehr2 (5’- CTC TAC ACT AGG AAT TCC GCT AT -3’), as described 161
elsewhere (Goodman et al., 1996). In addition, the presence of A. phagocytophilum was 162
excluded based on a specific real-time TaqMan assay (Wicki et al., 2000). Five samples were 163
further analyzed by conventional PCR with primers specific for the msp4 gene of A. ovis and 164
A. marginale (de la Fuente et al., 2001) using the same conditions as indicated above for the 165
16S rRNA gene of M. ovis. The PCR products were then sequenced.
166
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167
2.6. Statistical analyses 168
169
Calculations were performed with the software Analyse-it (Analyse-it Software Ltd., Leeds, 170
United Kingdom) for Microsoft Excel. Risk factors for haemoplasma infections were 171
evaluated using the Mann-Whitney U Test for continuous variables and the Fishers exact test 172
(cell frequencies ≤ 5) or Chi
2Test (cell frequencies > 5) for categorical variables. Differences 173
were regarded as significant at P < 0.05.
174 175
3. Results
176 177
3.1. Clinical outbreak and characteristics of affected animals 178
179
In July and August of 2006, 39 adult animals from a Hungarian sheep flock (flock A) showed 180
typical signs of eperythrozoonosis. All affected animals were 5 to 12 years of age. In their 181
blood smears two morphologically distinguishable moieties were visible: multiple small 182
forms characteristic of M. ovis and individual smaller or larger coccoid bodies (Fig. 1). A total 183
of 30 sheep died within 4 to 30 days after the onset of clinical signs despite initiation of 184
oxytetracyclin treatment at two- to four-day intervals. Both mortality (30 out of 546; 5.5%) 185
and lethality (30 out of 39; 77%) were significantly higher (P = 0.0124 and P < 0.0001, 186
respectively) as compared to the previous outbreak in the same flock in 1997 (mortality 2.2%:
187
9 out of 412, lethality 25.7%: 9 out of 35).
188
Merino sheep represented only 22% (120 out of 546) of flock A, whereas the 189
neighboring flock (flock B: never diseased) consisted exclusively of this breed (Table 1). The 190
remaining sheep in flock A were predominantly Ille de France and Bábolna Tetra crossbreeds.
191
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Significantly fewer sheep that had been affected by the disease (groups A1 and A2), were 192
born locally (38%: 15 out of 39) as compared to those in group A3 that demonstrated no 193
clinical signs (65%: 330 out of 507; P = 0.0016; Table 1). Among the group of sheep that had 194
died during the 2006 outbreak (A1), there were significantly fewer sheep born prior to the 195
1997 outbreak (33%: 10 out of 30) than in group A2, which consisted of the survivors of the 196
2006 disease outbreak (78%: 7 out of 9; P = 0.0262; Table 1).
197
Haematological values were within the normal range, except for haematocrit and 198
haemoglobin concentrations in the recently diseased sheep (group A2). These sheep had 199
significantly lower haematocrit (0.25 0.08) and haemoglobin (76.5 g/l 24.5 g/l) levels than 200
those in healthy control group A3, which exhibited values of (0.38
0.10; P = 0.0168) and 201
(105.1 g/l 13.5 g/l; P = 0.018), respectively. In group B the haematocrit was 0.3250.05 and 202
the haemoglobin concentration 100.2516.41 g/l.
203 204
3.2. Sample prevalence of ovine haemotropic mycoplasmas 205
206
Using a real-time PCR assay, 52% of the examined animals (17 out of 33) tested positive, 207
including sheep from all three sample groups: A2, A3, and B (Table 1). No significant 208
differences were found in sample prevalence among the three groups. All sheep that tested 209
positive by real-time PCR were also positive in conventional PCR and showed PCR products 210
of different lengths.
211 212
3.3. Molecular characterization of ovine haemotropic mycoplasmas 213
214
Cloning and sequencing of the near-complete 16S rRNA gene of haemotropic mycoplasmas 215
was successful for four sheep (one from group A2, one from A3, and two from flock B), with
216
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two to five clones obtained per sheep. Four sequences (designated IS-1 to IS-4) showed 99.4- 217
99.8% identity with M. ovis (AF338268). In contrast, five isolates (designated IS-5 to IS-9) 218
were found to be only 96.8-97.9% identical to M. ovis (Table 2). On a phylogenetical tree four 219
of the latter made up a separate cluster, while one occupied an intermediate position between 220
the M. ovis cluster and the new cluster (Fig. 2). The 16S rRNA gene sequence of the new 221
isolates differed from that of M. ovis (AF338268) at several nucleotide positions along the full 222
length of the gene (Table 2). In addition, a gap of 17 bp, including nucleotides 440 to 456, 223
was demonstrated in IS-5 to IS-9. Identical isolates were found in more than one animal, but 224
dissimilar isolates of both strains were also detected in the same animal (Table 2).
225 226
3.4. Co-infection with an Anaplasma sp.
227 228
Out of the 33 blood samples tested by molecular methods, 24 were found to be positive for an 229
Anaplasma sp., as determined by conventional PCR, but negative for A. phagocytophilum 230
based on specific real-time PCR. Sequencing results for part of the msp4 gene from five sheep 231
confirmed the presence of A. ovis (data not shown). Concomitant A. ovis infection was 232
significantly more prevalent in flock A (21 out of 25) than flock B (3 out of 8; P = 0.0201;
233
Table 1). Characteristic inclusion bodies could be observed by microscopy (Fig. 1).
234 235
4. Discussion
236 237
This is the first demonstration of the existence of two genetically distinct ovine haemotropic 238
mycoplasma strains.
239
Mycoplasma ovis infection is usually accompanied by mild clinical signs and no 240
fatalities among adult sheep (Neimark et al., 2004). This is in contrast to the outbreak reported
241
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in the present study. However, the prevalence of haemotropic mycoplasma infection in the 242
diseased flock (A) was not significantly different from that in flock B, where clinical signs 243
attributable to M. ovis have never been observed. Besides differences in breed composition of 244
the two flocks, the most important factor that could have influenced the clinical manifestation 245
is the co-infection with A. ovis. This turned out to be significantly less prevalent in the healthy 246
flock (B) than in the diseased flock (A). Although simultaneous eperythrozoonosis and 247
anaplasmosis have been documented (Splitter et al., 1955), no reports are currently available 248
on the potential synergism between these two haemotropic bacteria. Decreased haemoglobin 249
concentrations and haematocrit values reported for eperythrozoonosis (Gulland et al., 1987) 250
are usually not detected in A. ovis-infected adult sheep (Splitter et al., 1956; Hornok et al., 251
2007). Therefore, these changes detected in the present study were most likely caused by 252
haemotropic mycoplasmas.
253
In the unaffected group (A3) there was a higher proportion of locally born sheep than 254
in the two groups affected by M. ovis (A1, A2). This raises the possibility of a certain degree 255
of innate immunity (natural resistance) associated with endemic foci, which is known to exist 256
against other agents infecting erythrocytes (Gern et al., 1988). At the same time, sheep that 257
survived the current disease outbreak were more frequently born prior to the previous 258
outbreak in 1997 than sheep that succumbed, suggesting the development of an age-related 259
and/or acquired immunity to haemotropic mycoplasmas.
260
The disease outbreak between August and November of 1997 may have been a 261
consequence of the introduction of infected animals into the flock (Hajtós et al., 1998).
262
However, no animals were introduced into flock A in 2006. Therefore, exclusive 263
manifestation of the disease from July to August may have been associated with the seasonal 264
activity of potential arthropod vectors. Since mosquitoes may transmit M. ovis (Daddow, 265
1980; Howard, 1975) and they depend on water for their development, it may be relevant that
266
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the pastures grazed by sheep flocks A and B are flanked by a river. It is documented that 267
heavy rainfalls with floods in 2006 entailed an early summer mosquito invasion in Hungary 268
and promoted vector-borne transmission of different agents in the region (Vasconcelos, 269
2006), possibly including an upsurge in ovine haemoplasma infection of naive animals. On 270
the other hand, ticks as competent vectors of certain haemotropic mycoplasmas (Seneviratna 271
et al., 1973) were not found in the pastures or on the sheep in 2006 or 2007 (data not shown).
272
The disease outbreak in 2006 may have also been influenced by the appearance of the 273
second, genetically distinct ovine haemoplasma strain. Reclassification of haemotropic 274
Mycoplasma spp. was based on 16S rRNA gene sequences (Neimark et al., 2001).
275
Accordingly, the new sheep haemoplasma strain is most closely related to M. ovis (96.8- 276
97.9% identity to AF338268). At the same time, it is also substantially different from it, most 277
notably due to a 17 bp long deletion. Similar genotypic variants of haemoplasmas (with or 278
without a gap in their 16S rRNA sequence) were shown to exhibit higher or lower 279
pathogenicity in other hosts (Jensen et al., 2001; Neimark et al., 2001). The overall ratio of 280
16S sequence divergence between M. ovis and the novel ovine haemotropic mycoplasma 281
strain is close to the value for the declaration of a new species (Drancourt and Raoult, 2005).
282
However, to confirm if the novel strain deserves the status of a Candidatus, preferably 283
additional genes should be compared and the pathogenicity of the agent further evaluated in 284
monoinfections.
285
In conclusion, although M. ovis can be regarded as mildly pathogenic, concurrent 286
infection with divergent strains and/or other haemotropic bacteria may result in a more severe 287
clinical manifestation and even mortality among adult sheep.
288 289
Acknowledgements
290
291
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The authors would like to thank the invaluable help of J. Fodor, E. Gönczi, T. Meili, Prof. M.
292
Rusvai, A. Perreten and K. Museux. Laboratory work was performed with logistical support 293
from the Center for Clinical Studies at the Vetsuisse Faculty of the University of Zurich.
294
Regina Hofmann-Lehmann is the recipient of a Swiss National Science Foundation 295
professorship (grant numbers PP00B-102866/1 and PP00B—119136/1).
296 297
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363
Accepted Manuscript
Seneviratna, P., Weerasinghe, N., Ariyadasa, S., 1973. Transmission of Haemobartonella 364
canis by the dog tick, Rhipicephalus sanguineus. Res. Vet. Sci. 14, 112-114.
365 366
Splitter, E.J., Twiehaus, M.J., Castro, E.R.,1955. Anaplasmosis in sheep in the United States.
367
J. Am. Vet. Med. Assoc. 125, 244-245.
368 369
Splitter, E.J., Anthony, H.D., Twiehaus, M.J., 1956. Anaplasma ovis in the United States. Am.
370
J. Vet. Res. 17, 487-491.
371 372
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity 373
of progressive multiple sequence alignment through sequence weighting, position-specific 374
gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680.
375 376
Vasconcelos, P., 2006. Flooding in Europe: a brief review of the health risks. Euro. Surveill.
377
11, E060420.2. http://www.eurosurveillance.org/ew/2006/060420.asp#2 378
379
Wicki, R., Sauter, P., Mettler, C., Natsch, A., Enzler, T., Pusterla, N., Kuhnert, P., Egli, G., 380
Bernasconi, M., Lienhard, R., Lutz, H., Leutenegger, C.M., 2000. Swiss Army Survey in 381
Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia 382
phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus 383
in ticks. Eur. J. Clin. Microbiol. Infect. Dis. 19, 427-432.
384
385
Accepted Manuscript
Figure captions:
386 387
Figure 1. Blood smear of a sheep concomitantly infected with Anaplasma ovis (thin arrow) 388
and haemotropic mycoplasmas (thick arrow). The former are usually individually situated in 389
red blood cells (dark basophilic), whereas the latter tend to occur in multiplicity (clustered or 390
separated) on erythrocytes (therefore brightly basophilic).
391 392
Figure 2. Phylogenetic tree of the near-complete 16S rRNA gene sequences of haemotropic 393
mycoplasmas available in GenBank in comparison with those of the isolates from the 394
Hungarian sheep. The numbers at the nodes were generated from 1,000 Bootstrap 395
resamplings. The bar represents the mean number of differences per 50 sites.
396
397
Accepted Manuscript
Figure 1
Accepted Manuscript
novel ovine haemoplasma strain IS-5 novel ovine haemoplasma strain IS-7 novel ovine haemoplasma strain IS-6 novel ovine haemoplasma strain IS-8 novel ovine haemoplasma strain IS-9 Mycoplasma ovisIS-4
Mycoplasma ovisIS-2 Mycoplasma ovisIS-3 Mycoplasma ovisIS-1 Mycoplasma ovis (AF338268) Mycoplasma wenyonii (AY946266)
'Candidatus mycoplasma haemodidelphidis‘ (AF178676) 'Candidatus mycoplasma haemolamae‘ (AF306346)
Mycoplasma suis (AY492086)
'Candidatus mycoplasma haemoparvum‘ (AY532390) 'Candidatus mycoplasma haemominutum‘ (AY150980) 'Candidatus mycoplasma kahanei‘ (AF338269)
Mycoplasma haemomuris (U82963) Mycoplasma coccoides (AY171918)
'Candidatus mycoplasma turicensis‘ (DQ157152) 'Candidatus mycoplasma haemobovis‘ (EF616467)
Mycoplasma haemocanis (AY529641) Mycoplasma haemofelis (AY150984)
Mycoplasma pneumoniae (NC 000912) 94
96 100 88
46 87
84
100 99
97 59 63
87
100
100 100
74
50 96
Figure 2
Accepted Manuscript
Table 1. Data obtained for sheep in the two flocks (A and B) and results of molecular evaluation of their samples. Flock A consisted of 546
sheep which were divided into three groups (A1-3) according to the outcome in the 2006 disease outbreak.
Group Outcome in 2006 outbreak
Total number of sheep
Number (%) of merino
Number (%) born locally
Number (%) born before the
1997 outbreak
Number of samples for
PCR
Number (%) of haemoplasma- positive samples
Number (%) of anaplasma- positive samples
A1 Sick,
died
30 9 (30%) 13 (43% 10 (30%) n.a. n.a. n.a.
A2 Sick,
survived
9 3 (30%) 2 (22%) 7 (78%) 9 6 (67%) 7 (78%)
A3 Not
diseased
507 108 (27%) 330 (65%) 176 (35%) 16 7 (44%) 14 (88%)
B Never
diseased
545 545 (100%) n.a. n.a. 8 4 (50%) 3 (38%)
Abbreviation: n.a. – not available
Table 1
Accepted Manuscript
Table 2. Result of the alignment of the nearly complete 16S rRNA gene of nine ovine haemotropic mycoplasma isolates (IS-1 to IS-9) and their comparison with
the Mycoplasma ovis sequence. Upper index small letters next to the name of an isolate indicate animals from which it was obtained.
Position
1 101 118 119 125 139 188 218 224 272 288 319 340 395 440-456 507 510 514 635 648 673 690 701 753 763 953 1183 1274 1302 1337M. ovis
G T C G G G G G G G C G C
AG...GAC C G C G C A C G C G C C C T
IS-1
aA C * T * * * * * * * * * **...** * * * * * * * * * * * * T T A
IS-2
aA C * T * * * * * * * * * **...** * * * * * * * * * * A T T T A
IS-3
a,b,dA C * T * * * * * * * * * **...** * * * * * * * * * * * * * * *
IS-4
cA C * T * * * * * * * * * **...** * * * * * * * * * * * * * T A
IS-5
a* C T T * T A A A T T A T
...T T * T A T G A A T A T T T A
IS-6
a* C T T * T A A A T T A T
...T T * T A T G A * * A T T T A
IS-7
b* C T T A T A A A T T A T
...T T * T A T G A A T A T T T A
IS-8
c,d* * * T * T * A A T T A T
...T T T T A * G A * T * T T T A
IS-9
cA C * T * * * * * * * * *
...T T T T A * G A * T * * * * *
1
Position numbers given with respect to M. ovis (AF338268). Nucleotides identical to the latter are given as asterisks; mismatches are listed.
Upper indexes: a – one sheep from group A2; b – one sheep from group A3; c and d – two sheep from flock B
Table 2
